Method for transferring a substance to or from a closed system

ABSTRACT

The present invention relates to a cap which can form an essentially leak-proof seal with an open-ended vessel capable of receiving and holding fluid specimens or other materials for analysis. To minimize potentially contaminating contact between a fluid sample present in the vessel and humans or the environment, the present invention features a cap having a frangible seal which is penetrable by a plastic pipette tip or other fluid transfer device. The cap further includes filtering means for limiting dissemination of an aerosol or bubbles once the frangible seal has been pierced. The filtering means is positioned between the frangible seal and retaining means. The retaining means is positioned on the cap above the filtering means and may be used to contain the filtering means within the cap. The retaining means may be comprised of a material which is penetrable by a fluid transfer device.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional of U.S. application Ser. No. 10/093,511, filed Mar.8, 2002, now pending, the contents of which are hereby incorporated byreference herein, which claims the benefit of U.S. ProvisionalApplication No. 60/274,493, filed Mar. 9, 2001.

FIELD OF THE INVENTION

The present invention relates to caps for use in combination withfluid-holding vessels, such as those designed to receive and retainbiological specimens for clinical analysis, patient monitoring ordiagnosis. In particular, the present invention relates to a cap whichis penetrable by a fluid transfer device used to transfer fluids to orfrom a fluid-holding vessel, where the vessel and cap remain physicallyand sealably associated during a fluid transfer.

INCORPORATION BY REFERENCE

All references referred to herein are hereby incorporated by referencein their entirety. The incorporation of these references, standingalone, should not be construed as an assertion or admission by theinventors that any portion of the contents of all of these references,or any particular reference, is considered to be essential material forsatisfying any national or regional statutory disclosure requirement forpatent applications. Notwithstanding, the inventors reserve the right torely upon any of such references, where appropriate, for providingmaterial deemed essential to the claimed invention by an examiningauthority or court. No reference referred to herein is admitted to beprior art to the claimed invention.

BACKGROUND OF THE INVENTION

Collection devices are a type of cap and vessel combination commonlyused for receiving and storing biological specimens for delivery toclinical laboratories, where the specimens may be analyzed to determinethe existence or state of a particular condition or the presence of aparticular infectious agent, such as a virus or bacterial microorganism.Types of biological specimens commonly collected and delivered toclinical laboratories for analysis include blood, urine, sputum, saliva,pus, mucous and cerebrospinal fluid. Since these specimen-types maycontain pathogenic organisms, it is important to ensure that collectiondevices are constructed to be substantially leak-proof during transportfrom the site of collection to the site of analysis. This feature ofcollection devices is especially important when the clinical laboratoryand the collection facility are remote from one another, increasing thelikelihood that the collection device will be inverted or severelyjostled during transport and potentially subjected to substantialtemperature and pressure fluctuations.

To prevent specimen leakage, and possible contamination of thesurrounding environment, collection device caps are typically designedto be screwed, snapped or otherwise frictionally fitted or welded ontothe vessel component, thereby forming a substantially leak-proof sealbetween the cap and the vessel. In addition to preventing fluid specimenfrom leaking, a substantially leak-proof seal formed between the cap andthe vessel components of a collection device may also aid inameliorating exposure of the specimen to potentially contaminatinginfluences from the immediate environment. This aspect of a leak-proofseal is important for preventing the introduction of contaminants intothe collection device that could alter the qualitative or quantitativeresults of an assay.

While a leak-proof seal should prevent specimen seepage duringtransport, the actual removal of the cap from the vessel prior tospecimen analysis presents another potential opportunity forcontamination. When removing the cap, specimen which may have collectedon the underside of the cap during transport could come into contactwith a clinician, possibly exposing the clinician to a harmful pathogenpresent in the fluid sample. And if the specimen is proteinaceous ormucoid in nature, or if the transport medium contains detergents orsurfactants, then a film or bubbles could form around the mouth of thevessel during transport which could burst when the cap is removed fromthe vessel, thereby disseminating specimen into the testing environment.Another risk associated with cap removal is the potential for creating acontaminating aerosol which may lead to false positives or exaggeratedresults in other specimens being simultaneously or subsequently assayedin the same general work area through cross-contamination. It is alsopossible that specimen residue from one collection device, which mayhave been inadvertently transferred to a gloved hand of a clinician,will come into contact with specimen from another collection devicethrough routine or careless removal of caps and handling of thecollection devices.

Concerns with cross-contamination are especially acute when the assaybeing performed involves nucleic acid detection and includes anamplification procedure such as the well known polymerase chain reaction(PCR), or a transcription-based amplification system such astranscription-mediated amplification (TMA). (A review of severalamplification procedures currently in use, including PCR and TMA, isprovided in HELEN H. LEE ET AL., NUCLEIC ACID AMPLIFICATION TECHNOLOGIES(1997).) Since amplification is intended to enhance assay sensitivity byincreasing the quantity of targeted nucleic acid sequences present in aspecimen, transferring even a minute amount of pathogen-bearing specimenfrom one vessel, or target nucleic acid from a positive control sample,to another vessel containing an otherwise negative specimen could resultin a false-positive result.

To minimize the potential for creating contaminating specimen aerosols,and to limit direct contact between specimens and humans or theenvironment, it is desirable to have a collection device cap which canbe penetrated by a fluid transfer device (e.g., a pipette tip which canbe used with an air displacement pipette) while the cap remainsphysically and sealably associated with the vessel. The material andconstruction of the penetrable aspect of the cap should facilitate theventing of air displaced from the interior space of the collectiondevice to ensure accurate fluid transfers and to prevent a rapid releaseof aerosols as the fluid transfer device is being inserted into orwithdrawn from the collection device. And, because air is vented fromthe interior space of the collection device after the cap has beenpenetrated, it would be particularly helpful if means were included forminimizing aerosol release through the cap once it was penetrated by thefluid transfer device. Also, to limit the amount of potentiallycontaminating fluid present on the exterior of the fluid transfer deviceafter it is has been withdrawn from the collection device, it would beadvantageous if the cap also included means for wiping or absorbingfluid present on the outside of the fluid transfer device as it is beingwithdrawn from the collection device. To prevent damage to the fluidtransfer device which could affect its ability to predictably andreliably dispense or draw fluids, and to facilitate its use in manualpipetting applications, the cap should also be designed to limit theforces necessary for the fluid transfer device to penetrate the cap.Ideally, the collection device could be used in both manual andautomated formats and would be suitable for use with disposable pipettetips made of a plastic material.

Collection device caps which can be penetrated by a fluid transferdevice will have other advantages, as well, including the time-savingsresulting from clinicians not having to manually remove caps fromvessels before retrieving sample aliquots from the collection devicesfor assaying. Another advantage of penetrable collection device capswould be the reduction in repetitive motion injuries suffered byclinicians from repeatedly unscrewing caps.

SUMMARY OF THE INVENTION

The present invention solves the potential contamination problemsassociated with conventional collection devices by providing apenetrable cap for use with a vessel component of a collection devicewhich includes: (i) a closed side wall having an inner surface, an outersurface, a top surface and a bottom surface; (ii) attachment means forfixing the cap to an open end of the vessel in sealing engagement; (iii)a ledge which extends in a radial and inward direction from an innersurface of the side wall of the cap and has an end surface which definesan aperture sized to receive a fluid transfer device, where the innersurface of the side wall of the cap and a top surface of the ledgedefine a first bore; (iv) a frangible seal for preventing the passage ofa fluid from an interior space of the vessel into the first bore whenthe cap is fixed to the vessel in sealing engagement, where the seal isaffixed to either the top surface or a bottom surface of the ledge; (v)filtering means for impeding or preventing the release of an aerosol orbubbles from the interior space of the vessel to the atmosphere, wherethe filtering means is positioned substantially within the first bore;and (vi) retaining means for containing the filtering means within thefirst bore. (A “closed side wall”is one which lacks fully exposed endsurfaces.) The retaining means is preferably affixed to a top wall ofthe cap. The side wall, the flange and the ledge of the cap are moldedfrom a plastic material and preferably form a unitary piece.

In an alternative and preferred embodiment, the penetrable cap includesa skirt which depends from the bottom surface of the ledge, where aninner surface of the skirt defines a second bore having a diameter orwidth smaller than that of the first bore. The skirt may be included,inter alia, to further prevent a fluid from leaking from the interior ofthe vessel when the cap is fixed to the vessel in sealing engagement.(By “sealing engagement”is meant touching contact between solid surfaceswhich is intended to prevent or impede the passage of a fluid.) An outersurface of the skirt preferably includes a seal bead which is infrictional contact with an inner surface of the vessel. With thisembodiment, the frangible seal may be affixed to either the top surfaceof the ledge or to a bottom surface of the skirt. The side wall, theflange, the ledge and the skirt of the cap of this embodiment are moldedfrom a plastic material and preferably form a unitary piece.

In another embodiment of the present invention, the retaining means is asecond frangible seal. The second frangible seal may comprise the sameor a different material than the frangible seal affixed to the top orbottom surface of the ledge, or to the bottom surface of the skirt. Bothseals are penetrable by a fluid transfer device with the application ofmoderate manual force and each seal preferably comprises a foil.

In still another embodiment of the present invention, the retainingmeans comprises a foil ring having a centrally located hole which issized to receive a fluid transfer device and which is substantiallyaxially aligned with the first bore and the second bore, if present. Thediameter or width of this hole is smaller than the diameter or width ofa filter contained within the first bore so that the foil ring canfunction to contain the filter within the cap. The foil ring of thisembodiment may be affixed to the top wall of the cap by means of anadhesive or by means of a plastic liner which has been applied to thefoil ring and which can be welded to the surface of the top wall of thecap.

In yet another embodiment of the present invention, the retaining meansincludes a plastic disc having a hole formed therein which is sized toreceive a fluid transfer device and which is substantially axiallyaligned with the first bore and the second bore, if present. Theretaining means of this embodiment functions to contain a filter withinthe first bore. The disc may be affixed to the top wall of the cap orthe top wall may be adapted to include a seat for receiving the disc in,for example, a frictional or snap fit.

In a further embodiment, the retaining means comprises a removable sealwhich is designed to limit exposure of a filter to environmentalcontaminants and may include a tab for easy removal prior to penetratingthe cap. Because this seal may be removed prior to penetration of thecap, it is not a requirement that this particular retaining means becomprised of a frangible material which can be pierced by a fluidtransfer device applying moderate manual force. Since the removable sealcan function to protect the filter against external contaminants duringshipping, the removable seal may be applied, for example, to the fixeddisc described above for retaining the filter within the first bore.

In still another embodiment, the cap is provided as part of a collectiondevice which includes a vessel for containing fluids. When provided as apart of a collection device, the cap preferably includes the skirtfeature described above, which is positioned adjacent to an innersurface of an open end of the vessel to impede the passage of a fluidfrom the interior space of the vessel to the environment outside of thecollection device. Including a seal bead on an outer surface of theskirt further facilitates this objective by increasing the pressurewhich is exerted by the skirt on the inner surface of the vessel. Thecollection device may contain, for example, a dry powder, pellets ofchemical reagents, buffers, stabilizers, or a transport medium forpreserving a specimen while it is being shipped from a collectionlocation to a site for analysis. The collection device may also beprovided in packaged combination with a specimen retrieval device (e.g.,a swab) for obtaining a specimen from a human, animal, water,environmental, industrial, food or other source. Instructional materialsmay additionally be included with the collection device which detailproper use of the collection device when obtaining or transporting aspecimen or appropriate techniques for retrieving a fluid sample fromthe collection device at the site of analysis. When in packagedcombination, the recited items are provided in the same container (e.g.,a mail or delivery container for shipping), but do not need to be per sephysically associated with one another in the container or combined inthe same wrapper or vessel within the container.

In yet another embodiment, the cap can be used in a method forretrieving a fluid substance from the vessel component of a collectiondevice with a plastic pipette tip for use with an air displacementpipette. When the cap is penetrated by the pipette tip, air passagewaysare formed between the pipette tip and the frangible seal or seals ofthe cap, thereby facilitating the venting of air from within the vessel.After fluid is removed from the collection device, at least some portionof the fluid sample may be exposed to amplification reagents andconditions permitting a targeted nucleic acid sequence which may bepresent in the fluid sample to be amplified. Various amplificationprocedures, and their associated reagents and conditions, are well knownto those skilled in the art of nucleic acid diagnostics.

In still another embodiment, a method is provided for removing a fluidsubstance contained in a closed system comprising a cap and afluid-holding vessel. In addition to the cap and vessel components, thephrase “closed system” is used herein to refer to a cap that is fixed toa vessel in sealing engagement to prevent the fluid contents of thesystem from escaping into the surrounding environment. The methodincludes penetrating first and second frangible seals affixed to the capwith a fluid transfer device, where the second seal is axially alignedbelow the first seal. Penetration of the first and second seals by thefluid transfer device results in the formation of air passagewaysbetween the seals and the fluid transfer device which aid in venting airfrom the interior space of the system. The fluid transfer device ispreferably a plastic pipette tip for use with an air displacementpipette. In a preferred mode, the method further includes passing thefluid transfer device through a filter contained within the cap andinterposed between the first and second seals.

Once fluid has been removed from the system in this method, some or allof the fluid sample may be exposed to amplification reagents andconditions permitting a targeted nucleic acid sequence which may bepresent in the fluid sample to be amplified. As noted above, a varietyof amplification procedures are well known to those skilled in the artof nucleic acid diagnostics, and appropriate reagents and conditions foruse with any of these amplification procedures could be determinedwithout engaging in undue experimentation.

In yet a further embodiment of the present invention, a method isprovided for depositing substances into the collection devices and otherclosed systems of the present invention by means of a substance transferdevice capable of transporting fluids (e.g., specimen or assay reagents)or solids (e.g., particles, granules or powders). This embodiment of thepresent invention is particularly useful for diagnostic assays which canbe performed in a single reaction vessel and where it is desirable tomaintain the contents of the vessel in a substantially closedenvironment. The steps of this embodiment are similar to those of otherpreferred methods described herein, except that one or more substanceswould be deposited into the vessel component rather than retrieving afluid sample contained therein.

The methods of the presently claimed invention may be performed manuallyor adapted for use with a semi-automated or fully automated instrument.Examples of instrument systems which could be readily adapted for usewith the collection devices or other closed systems of the presentinvention include the DTS™ 400System (detection only) and the DTS™1600System (amplification and detection) (Gen-Probe Incorporated; SanDiego, Calif.). See Acosta et al., “Assay Work Station,”U.S. Pat. No.6,254,826. Other fully automated instrument systems are disclosed byAmmann et al., “Automated Process for Isolating and Amplifying a TargetNucleic Acid Sequence,” U.S. Pat. No. 6,335,166.

These and other features, aspects, and advantages of the presentinvention will become apparent to those skilled in the art afterconsidering the following detailed description, appended claims andaccompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an exploded perspective view of the cap and vessel componentsof a preferred collection device of the present invention.

FIG. 2 is an enlarged top plan view of the core cap of FIG. 1.

FIG. 3 is an enlarged bottom view of the core cap of FIG. 1.

FIG. 4 is an enlarged partial section side view of the collection deviceof FIGS. 1-3 (showing core cap only), taken along the 4-4 line thereof.

FIG. 5 is an enlarged partial section side view of another collectiondevice according to the present invention.

FIG. 6 is an enlarged section side view of the cap of FIG. 1.

FIG. 7 is an enlarged section side view of another cap according to thepresent invention.

FIG. 8 is an enlarged section side view of a frangible seal according tothe present invention.

FIG. 9 is an enlarged section side view of another frangible sealaccording to the present invention.

FIG. 10 is an enlarged top plan view of the core cap and filter of FIG.1.

FIG. 11 is a top plan view of the cap of FIGS. 6 and 7 showingperforations in the frangible seal.

FIG. 12 is an enlarged top plan view of a further cap according to thepresent invention.

FIG. 13 is an enlarged section side view of yet another cap according tothe present invention.

FIG. 14 is a top plan view of the cap of FIG. 13.

FIG. 15 is an enlarged section side view of still another cap accordingto the present invention.

FIG. 16 is a top plan view of the cap of FIG. 15.

FIG. 17 is a partial section side view of the collection device of FIG.1, after it has been penetrated by a fluid transfer device.

FIG. 18 is a top plan view of the cap and fluid transfer device of FIG.17.

DETAILED DESCRIPTION OF THE INVENTION

While the present invention may be embodied in a variety of forms, thefollowing description and accompanying drawings are merely intended todisclose some of these forms as specific examples of the presentinvention. Accordingly, the present invention is not intended to belimited to the forms or embodiments so described and illustrated.Instead, the full scope of the present invention is set forth in theappended claims.

With reference to the figures, preferred caps 30A-E of the presentinvention are shown alone or in combination with a vessel 20 which canbe used for receiving and storing fluid specimens for subsequentanalysis, including analysis with nucleic acid-based assays orimmunoassays diagnostic for a particular pathogenic organism. When thedesired specimen is a biological fluid, the specimen can be, forexample, blood, urine, saliva, sputum, mucous or other bodily secretion,pus, amniotic fluid, cerebrospinal fluid or seminal fluid. However, thepresent invention also contemplates materials other than these specificbiological fluids, including, but not limited to, water, chemicals andassay reagents, as well as solid substances which can be dissolved inwhole or in part in a fluid milieu (e.g., tissue specimens, stool,environmental samples, food products, powders, particles and granules).The vessel 20 is preferably capable of forming a substantiallyleak-proof seal with the cap 30A-E and can be of any shape orcomposition, provided the vessel is shaped to receive and retain thematerial of interest (e.g., fluid specimen or assay reagents). Where thevessel 20 contains a specimen to be assayed, it is important that thecomposition of the vessel be essentially inert so that it does notsignificantly interfere with the performance or results of an assay. Apreferred vessel 20 is formed of polypropylene and has a generallycylindrical shape which measures approximately 13mm×82 mm.

As illustrated in the figures, particularly preferred caps 30A-E of thepresent invention include an integrally molded core structure 31A(referred to herein as the “core cap”) which comprises: (i) a generallycylindrical side wall 35; (ii) a flange 36 depending from a bottomsurface 37 of the side wall and having an inner surface 38 adapted togrip an outer surface 21 of a generally cylindrical side wall 22 of anopen-ended vessel 20; (iii) a ledge 39 extending radially inwardly froman inner surface 40 of the side wall 35 above the flange 36; and (iv) agenerally cylindrical skirt 41 depending from a bottom surface 42 of theledge in a substantially parallel orientation to the flange. The innersurface 40 of the side wall 35 and a top surface 43 of the ledge 39define a first bore 44, as shown in FIG. 4, which is sized to receive afilter 33, as shown in FIGS. 6 and 7, that may be frictionally fitted orotherwise immobilized within the first bore. In a preferred embodiment,the ledge 39 aids in retaining the filter 33 within the first bore 44during penetration of the cap 30A-E by a fluid transfer device. Theledge 39 can also function as a surface for affixing a frangible seal32, as depicted in FIG. 6. An inner surface 45 of the skirt 41 beneaththe top surface 43 of the ledge 39 defines a second bore 46 which issmaller in diameter than the first bore 44 and is sized to permitmovement therethrough of a fluid transfer device. (The proximal portionof the skirt 41, where the top surface 43 of the ledge 39 meets theinner surface 45 of the skirt, may be chamfered to deflect a misalignedfluid transfer device during penetration of the cap 30A-E, providedsufficient surface area remains on the top surface of the ledge foraffixing the frangible seal 32 thereto.) As shown in FIG. 7, the skirt41 includes a bottom surface 47 which may serve as an alternate locationfor affixing the frangible seal 32.

In an alternative core cap 31B embodiment shown in FIG. 5, the skirt 41is eliminated from the core cap 31A structure shown in FIG. 4. In thisembodiment, the frangible seal 32 may be affixed to either the bottomsurface 42 or the top surface 43 of the ledge 39. However, since theskirt 41 aids in preventing fluids from leaking from a collection device10, it may be desirable to include an alternative fluid retainer forthis core cap 31B embodiment, such as a neoprene O-ring (not shown)fitted between the bottom surface 37 of the side wall 35 and an annulartop surface 23 of the vessel 20.

While the ledge 39 of the core cap 31B shown in FIG. 5 forms a flangestructure having bottom and top surfaces 42, 43, this embodiment may bemodified so that the inner surface 40 of the side wall 35 is extendedradially inward until an end surface 62 of the ledge and the innersurface of the side wall are co-extensive. In this modified form of thecore cap 31B (not shown), the ledge 39 is defined by the bottom surface37 of the side wall 35, since the top surface 43 of the ledge iseliminated. Because the bore 44 of this embodiment is defined solely bythe inner surface 40 of the side wall 35, the frangible seal 32 must beaffixed to the bottom (or sole) surface 42 of the ledge 39.

A similar modification may be made to the preferred core cap 31A,whereby the inner surface 40 of the side wall 35 is extended radiallyinward until the inner surface 45 of the skirt 41 is co-extensive withthe inner surface of the side wall. This modified form of the core cap31A (not shown) eliminates the ledge 39, transforms the first and secondbores 44, 46 into a single bore, and requires that the frangible seal 32be affixed to the bottom surface 47 of the skirt 41. The disadvantage ofthese modified forms of the core caps 31A, 31B is that the alteration orelimination of the ledge 39 makes it is more difficult to maintain thefilter 33 within the cap 30A-E when the cap is penetrated by a fluidtransfer device. This problem may be overcome by adhering the filter 33to the side wall 35 of the cap 30A-E, the frangible seal 32 or theretaining means 34A-C, for example.

The core cap 31A-B may be integrally molded from a number of differentpolymer and heteropolymer resins, including, but not limited to,polyolefins (e.g., high density polyethylene (“HDPE”), low densitypolyethylene (“LDPE”), a mixture of HDPE and LDPE, or polypropylene),polystyrene, high impact polystyrene and polycarbonate. A currentlypreferred material for forming the core cap 31A-B is an HDPE materialsold under the tradename Alathon M5370 by GE Polymerland ofHuntersville, N.C. Skilled artisans will readily appreciate that therange of acceptable cap resins will, in part, depend upon the nature ofthe resin used to form the vessel, since the properties of the resinsused to form these two components will affect how well the cap 30A-E andvessel 20 components of a collection device 10 can form a leak proofseal and the ease with which the cap can be securely screwed onto thevessel. As with the vessel 20 component, the material of the core cap31A-B should be essentially inert with respect to a fluid substance(including assay reagents) contained in the collection device 10 so thatthe material of the core cap does not significantly interfere with theperformance or results of an assay.

The core cap 31A-B is injection molded as a unitary piece usingprocedures well-known to those skilled in the art of injection molding.After the core cap 31A-B has been formed and cured for a sufficientperiod of time, the following components are added to the core cap inthe indicated manner and in any practicable order: (i) the frangibleseal 32 to the top surface 43 of the ledge 39 of either core cap 31A-B,to the bottom surface 42 of the ledge of the alternative core cap 31B,or to the bottom surface 47 of the skirt 41 of the preferred core cap31A; (ii) a filter 33 within the first bore 44; and (iii) a retainer34A-D to the annular top wall 48.

The frangible seal 32 is included to provide a substantially leak-proofbarrier between the fluid contents of a collection device 10 and thefilter 33 contained in the first bore 44. For this reason, it is notcritical whether the frangible seal 32 is affixed to a surface 42, 43 ofthe ledge 39 or to the bottom surface 47 of the skirt 41. According to apreferred embodiment of the present invention, the width of the annulartop surface 43 of the ledge 39 is about 0.08 inches (2.03mm), thethickness of the ledge (the distance between the top and bottom surfaces43, 42 of the ledge) is about 0.038 inches (0.97 mm), the combined widthof the annular bottom surface 42 of the ledge and the exposed bottomsurface 37 of the side wall 35 is about 0.115 inches (2.92 mm), and thewidth of the annular bottom surface 47 of the skirt 41 is about 0.025inches (0.635 mm). The dimensions of these features of the core cap31A-B may vary, of course, provided sufficient surface area exists foraffixing the frangible seal 32 to the core cap in a substantiallyleak-proof manner.

The frangible seal 32 may be made of a plastic film (e.g., thinmonoaxially or biaxially oriented plastic film) or, preferably, of afoil (e.g., aluminum foil or other foil exhibiting low water vaportransmission), which can be affixed to a surface 42, 43 of the ledge 39or to the bottom surface 47 of the skirt 41 by means well known to thoseskilled in the art, including adhesives. The frangible seal 32 ispreferably not an integral component of the core cap 31A-B. If thefrangible seal 32 comprises a foil, it may further include acompatibilizer, such as a thin veneer of plastic applied to one or bothsurfaces of the foil, which will promote a substantially leak-proofattachment of the frangible seal to a surface of the core cap 31A-B withthe application of thermal energy. A heat sealer or heat inductionsealer may be used to generate the requisite thermal energy. (To avoidthe potentially deleterious effects of corrosion, it is recommended thatall portions of a metallic frangible seal 32 which might become exposedto the fluid contents of a collection device 10 during routine handlingbe coated with a plastic liner.) A TOSS Machine heat sealer (PackworldUSA; Nazareth, Pa.; Model No. RS242) is preferred for attaching thefrangible seal 32 to a surface 42,43 of the ledge 39 or to the bottomsurface 47 of the skirt 41. Ultrasonic and radio frequency weldingprocedures known to those skilled in the art may also be used to affixthe frangible seal 32 to the core cap 31A-B.

To further promote attachment of the frangible seal 32 to the core cap31A-B, a surface 42, 43 of the ledge 39 or the bottom surface 47 of theskirt 41 may be modified during injection molding of the core cap toinclude an energy director, such as an annular ring or series ofprotuberances. By limiting contact between the frangible seal 32 and aplastic surface of the ledge 39 or skirt 41, an energy director allowsthe frangible seal 32 to be affixed to the ledge or skirt in less timeand using less energy than would be required in its absence when anultrasonic welding procedure is followed. This is because the smallersurface area of the protruding energy director melts and forms a weldwith the plastic material of the frangible seal 32 more quickly than ispossible with a flat, unmodified plastic surface. An energy director ofthe present invention is preferably a continuous surface ring which istriangular in cross-section.

In order to facilitate the venting of air from within a collectiondevice 10, the frangible seal 32 is preferably constructed so that ittears when the seal is penetrated by a fluid transfer device, therebyforming air passageways 70 between the seal and the fluid transferdevice, as described in detail infra. To achieve this tearing, the seal32 preferably includes a brittle layer comprised of a hard plasticmaterial, such as a polyester. (See CHARLES A. HARPER, HANDBOOK OFPLASTICS, ELASTOMERS, AND COMPOSITES § 1.7.13 (1997 3d ed.) for adiscussion of the properties of polyesters.) As shown in FIG. 8, thepreferred seal 32 of the present invention is a co-laminate thatincludes a foil layer, a heat seal layer and an intervening brittlelayer (Unipac; Ontario, Canada; Product No. SG-75M (excluding the pulpboard and wax layers typically included with this product)). With thispreferred seal 32, the foil layer is an aluminum foil having a thicknessof about 0.001 inches (0.0254 mm), the heat seal layer is a polyethylenefilm having a thickness of about 0.0015 inches (0.0381 mm), and thebrittle layer is a polyester having a thickness of about 0.0005 inches(0.0127 mm). Because this particular seal 32 design would have ametallic surface exposed to the contents of a collection device 10 aftersealing—if applied to the bottom surface 42 of the ledge 39 or to thebottom surface 47 of the skirt 41—it is preferred that this seal 32 beapplied to the top surface 43 of the ledge, as shown in FIGS. 6, 13, 15and 17. In this way, the cap 30A, C, D and E of these embodiments willhave no metallic surfaces exposed to the fluid contents of an associatedvessel 20. While the diameter of the seal 32 will depend upon thedimensions of the cap 30A-E, the presently preferred seal has a diameterof about 0.5 inches (12.70 mm).

An alternative frangible seal 32 embodiment is depicted in FIG. 9, whichshows a top foil layer with a lower combined brittle/heat seal layercomprised of an epoxy resin. The epoxy resin is selected for itsmechanical strength, which will promote formation of the desired airpassageways 70 discussed above when the material is penetrated by afluid transfer device. And, so that this seal 32 can be affixed to aplastic surface using a commonly practiced thermoplastic weldingprocedure, the epoxy layer further includes a compatibilizer dispersedwithin the epoxy resin, as shown in FIG. 9. A preferred compatibilizerof this seal 32 embodiment is a polyethylene.

As illustrated in the figures, the filter 33 is positioned within thefirst bore 44 above the ledge 39 and is incorporated to retard or blockthe movement of an escaping aerosol or bubbles after the seal has beenpierced by a fluid transfer device. The filter 33 can also beconstructed to perform a wiping action on the outside of a fluidtransfer device as the fluid transfer device is being removed from acollection device 10. In a preferred mode, the filter 33 functions todraw fluids away from the outside of a fluid transfer device by means ofcapillary action. As used herein, however, the term “filter” refersgenerally to a material which performs a wiping function to removefluids present on the outside of a fluid transfer device and/or anabsorbing function to hold or otherwise sequester fluids removed fromthe outside of a fluid transfer device. For reasons discussed below, thefilters 33 of the present invention are composed of a material orcombination of materials having pores or interstices which admit thepassage of a gas. Examples of filter 33 materials which may be used withthe cap 30A-E of the present invention include, but are not limited to,pile fabrics, sponges, foams (with or without a surface skin), felts,sliver knits, a GORE-TEX® fabric, a fabric containing a LYCRA® fiber,and other materials and blends, both natural and synthetic. Thesematerials may also be mechanically or chemically treated to furtherimprove the intended functions of the filter 33. For example, nappingmay be used to increase the surface area and, therefore, the fluidholding capacity of a filter 33. The material of the filter 33 may alsobe pre-treated with a wetting agent, such as a surfactant, to lower thesurface tension of a fluid present on an outer surface of a fluidtransfer device. An acrylic binder might be used, for example, toactually bind the wetting agent to the filter 33 material. Additionally,the filter 33 may include a super-absorbent polymer, (see, e.g.,Sackmann et al., “Pre-Formed Super Absorbers with High SwellingCapacity,” U.S. Pat. No. 6,156,848), to prevent a fluid from escapingfrom a penetrated collection device 10.

To limit the unobstructed passage of air from within a collection device10 to the environment, the filter 33 is preferably made of a resilientmaterial whose original shape is restored, or substantially restored, asa fluid transfer device is being removed from the collection device.This characteristic of the filter 33 is especially important when thefluid transfer device has a non-uniform diameter, as is the case withmost pipette tips used with standard air displacement pipettes. Thus,materials such as pile fabric, sponges, foams, and fabrics containingLYCRA® fibers are preferred because they tend to quickly restore theiroriginal shape after exposure to compressive forces. Pile fabric is aparticularly preferred filter 33. An example of a preferred pile fabricis an acrylic material having a thickness of about 0.375 inches (9.53mm) which is available from Roller Fabrics of Milwaukee, Wis. as PartNo. ASW112. Examples of other acceptable pile fabrics include those madeof acrylic and polyester materials and which range in size from about0.25 inches (6.35 mm) to about 0.3125 inches (7.94 mm). Such pilefabrics are available from Mount Vernon Mills, Inc. of LaFrance, S.C. asPart Nos. 0446, 0439 and 0433. The filter 33 material is preferablyinert with respect to a fluid substance contained within the vessel 20.

Because the filter 33 is intended to remove fluid from the exterior of afluid transfer device and to capture fluid in the form of an aerosol orbubbles, it is best if the material and dimensions of the filtermaterial are chosen so that the filter does not become saturated withfluid during use. If the filter 33 does become saturated, fluid may notbe adequately wiped from the exterior of the fluid transfer device andbubbles may be produced as the fluid transfer device passes through thefilter or as air is displaced from within the collection device 10.Thus, it is important to adapt the size and adsorptive properties of thefilter 33 in order to achieve adequate wiping and aerosol or bubblecontainment. Considerations when selecting a filter 33 will include thecap configuration, the shape and size of the fluid transfer device andthe nature and amount of fluid substance contained in the vessel 20,especially in view of the number of anticipated fluid transfers for agiven collection device 10. As the amount of fluid a filter 33 is likelyto be exposed to increases, the volume of the filter material or itsabsorptive properties may need to be adjusted so that the filter doesnot become saturated during use.

It is also important that the filter 33 be constructed and arranged inthe cap 30A-E so that the flow of air out of the collection device 10remains relatively unimpeded as the cap is being penetrated by a fluidtransfer device. In other words, the material of the filter 33 and itsarrangement within the cap 30A-E should facilitate the venting of airdisplaced from within the collection device 10. Of course, this ventingproperty of the filter 33 needs to be balanced with the requirement thatthe filter material have sufficient density to trap an escaping aerosolor bubbles. Consequently, those skilled in the art will appreciate theneed to select or design filter 33 materials having matrices that arecapable of trapping an aerosol or bubbles, while simultaneouslypermitting air to be vented from within the collection device 10 oncethe underlying seal 32 has been pierced by a fluid transfer device.

As the figures show, the filter 33 is preferably sized to fit within thefirst bore 44 beneath the horizontal plane of the annular top wall 48.In a preferred cap 30A-E of the present invention, the filter 33 alsorests substantially or completely above the ledge 39, even though theseal 32 may be affixed to the bottom surface 47 of the skirt 41, asillustrated in FIG. 7. To better ensure that the filter 33 is notsubstantially moved from its position within the first bore 44 byfrictional contact with a fluid transfer device penetrating or beingremoved from the cap 30A-E, the filter may be bound to the top surface43 of the ledge 39 or to the inner surface 40 of the side wall 35 usingan inert adhesive. Notwithstanding, the filter 33 is preferably a pilefabric which is snugly fitted in the first bore 44 and retained there bymeans of the seal 32 and the retainer 34A-D, without the use of anadhesive. In preferred cap 30A-E embodiments, the first bore 44 is about0.50 inches (12.70 mm) in diameter and has a height of about 0.31 inches(7.87 mm).

The material and configuration of the filter 33 should be such that itcreates minimal frictional interference with a fluid transfer device asit is being inserted into or withdrawn from the collection device 10. Inthe case of a sponge or foam, for example, this may require boring ahole or creating one or more slits in the center of the filter 33 (notshown) which are sized to minimize frictional interference between thefilter and a fluid transfer device, while at the same time providingenough interference so that aerosol or bubble transmission is limitedand the wiping action is performed by the filter material. If a pilefabric is employed as the filter 33, the pile fabric is preferablyarranged in the manner shown in FIG. 10, such that the free ends ofindividual fibers (shown as squiggles, but not specifically identifiedwith a reference number) are oriented radially inwardly toward thelongitudinal axis 80 of the cap 30A-E and away from the pile fabricbacking 49 which is in touching or fixed contact with the inner surface40 of the side wall 35. When rolling the pile fabric for insertion intothe first bore 44, care should be taken not to wind the pile fabric sotightly that it will create excessive frictional interference with afluid transfer device penetrating the cap 30A-E, thereby substantiallyimpeding movement of the fluid transfer device. A particularly preferredpile fabric is available from Mount Vernon Mills, Inc. as Part No. 0446,which has a thickness of about 0.25 inches (6.35 mm) and is cut to havea length of about 1.44 inches (36.58 mm) and a width of about 0.25inches (6.35 mm).

To immobilize the filter 33 within the first bore 44, the caps 30A-E ofthe present invention include a retainer 34A-D positioned above thefilter, preferably on the annular top wall 48. In a preferred embodimentshown in FIGS. 6 and 7, the retainer 34A is a solid, generally circularfrangible seal which may be of the same or a different material than thefrangible seal 32 positioned beneath the filter 33. Preferably, theretainer 34A includes the same materials as the preferred seal 32described above, which comprises an aluminum foil layer, a polyesterbrittle layer, and a polyethylene heat seal layer (Unipac; Ontario,Canada; Product No. SG-75M (excluding the pulp board and wax layerstypically included with this product)). This retainer 34A can be appliedto the annular top wall 48 with a heat sealer or heat induction sealerin the same manner that the seal 32 is applied to a surface 42, 43 ofthe ledge 39 or to the bottom surface 47 of the skirt 41. Like thepreferred seal 32, the preferred retainer 34A has a foil layer thicknessof about 0.001 inches (0.0254 mm), a brittle layer thickness of about0.0005 inches (0.0127 mm), and a heat seal layer thickness of about0.0015 inches (0.0381 mm). The diameter of the preferred retainer isabout 0.625 inches (15.88 mm). Of course, the diameter of this preferredretainer 34A may vary and will depend upon the dimensions of the annulartop wall 48.

As illustrated in FIG. 11, the retainer 34A may be adapted to facilitatepenetration by including one or more series of perforations 50 whichextend radially outwardly from a center point 51 of the retainer. Thecenter point 51 of these radiating perforations 50 is preferablypositioned to coincide with the expected entry point of a fluid transferdevice. Also contemplated by the present invention are other types ofadaptations that would reduce the tensile strength of the retainer 34A,including creases, score lines or other mechanical impressions appliedto the material of the retainer. The same adaptations may also be madeto the seal 32, provided the seal will continue to exhibit low watervapor transmission characteristics after the collection device 10 isexposed to normal shipping and storage conditions.

Besides providing a means for keeping the filter 33 fixed within thefirst bore 44 prior to and during a fluid transfer, a seal retainer 34Acan protect the underlying filter from external contaminants prior topenetration of the cap 30A. Moreover, a cap 30A designed to completelyseal the filter 33 within the first bore 44 may be sterilized prior touse by, for example, gamma irradiation. Additionally, the retainer 34 ofsuch a cap 30A could be wiped with a disinfectant or the entirecollection device 10 could be irradiated with ultraviolet light prior topenetration to facilitate a sterile fluid transfer.

In those instances where the potential presence of contaminants on thefilter would not be of significant concern, however, the retainer maycomprise a foil ring, for example, which includes a centrally locatedhole which is sized to receive a fluid transfer device. As illustratedin FIG. 12, a cap 30C having a retainer 34B with a centrally locatedhole 52 could aid in retaining the filter 33 within the first bore 44,while at the same time limiting the number of surfaces that a fluidtransfer device would have to pierce in order to fully penetrate thecap. To retain the filter 33 within the first bore 44, the diameter ofthe hole 52 would have to be smaller than the diameter of the filterwhen the hole and the filter are substantially axially aligned.

Another cap 30D embodiment is illustrated in FIGS. 13 and 14. Theretainer 34C of this cap 30D is a plastic disc which includes acentrally located hole 53 sized to receive a fluid transfer device. Thedisc 34C may be affixed to the annular top wall 48 by means of anadhesive or welded by heat, ultrasound or other appropriate weldingmethod known to skilled artisans. Alternatively, the annular top wall 48may be modified to include a seat 54 which is sized to receive the disc34C in, for example, a frictional or snap fit. While this particular cap30D embodiment does not provide the filter 33 with a completely sealedenvironment, the disc retainer 34C can nevertheless function to containthe filter 33 within the first bore 44 during transport of thecollection device 10, as well as during a fluid transfer. If it isimportant to protect the filter 33 from potential contaminants prior touse, then the cap 30D of this embodiment could further include a seal,such as the frangible seal 34A described supra, affixed to a top 55 or abottom surface 56 of the disc 34C so that the hole 53 is fully andsealably covered. As illustrated in FIGS. 15 and 16, one such seal 34Dcould include a tab 57 for easy removal. With this design, the seal 34Dof a cap 30E could be removed just before penetration of the cap with afluid transfer device, allowing the filter 33 to be protected fromexternal contaminants immediately prior to use. An advantage of this cap30E over, for example, the cap 30A-B embodiments shown in FIGS. 6 and 7is that penetration of the cap will require less force since there isonly one seal 32, as opposed to the two seals 32, 34A of thoseembodiments, that the fluid transfer device needs to pierce.

When a cap 30A-E of the present invention is pierced by a fluid transferdevice 90 which is used to retrieve at least a portion of a fluid sample100 contained in a collection device 10, as shown in FIG. 17, one ormore tears are preferably formed in the frangible seal 32 and, ifcomprised of a frangible seal, the retainer 34A. As FIG. 18 illustrates,these tears in the frangible seal form air passageways 70 whichfacilitate the venting of air displaced from within a collection device10 as the fluid transfer device 90 enters the interior space 11 (definedas the space below the cap 30A-E and within inner surfaces 24, 25 of theside wall 22 and a bottom wall 26 of the vessel 20) of the collectiondevice 10. By providing means for venting air displaced from within acollection device 10, the volume accuracy of fluid transfers (e.g.,pipetting) will likely be improved. While a variety of fluid transferdevices can be used with the present invention, including hollow metalneedles and conventional plastic pipette tips having beveled or flattips, a preferred fluid transfer device is the Genesis series 1000 μlTecan-Tip (with filter), available from Eppendorf-Netherler-Hinz GmbH ofHamburg, Germany as Part No. 612-513. Fluid transfer devices of thepresent invention are preferably able to penetrate the frangible seal 32with the application of less than about 3 pounds force (13.34 N), morepreferably less than about 2 pounds force (8.90 N), even more preferablyless than about 1 pound force (4.45 N), and most preferably less thanabout 0.5 pounds force (2.22 N).

The insertion force, which is the total or additive force required topierce all penetrable surfaces of a cap 30A-E according to the presentinvention (i.e., the frangible seal 32, the filter 33 and, optionally,the retainer 34A) with a fluid transfer device, is preferably less thanabout 8 pounds force (35.59 N), more preferably less than about 6.5pounds force (28.91 N), even more preferably less than about 5 poundsforce (22.24 N), and most preferably less than about 4.5 pounds force(20.02 N). The withdrawal force, which is the force required tocompletely withdraw a fluid transfer device from the collection device10 after the cap 30A-E has been completely penetrated, is preferablyless than about 4 pounds force (17.79 N), more preferably less thanabout 3 pounds force (13.34 N), even more preferably less than about 2pounds force (8.90 N), and most preferably less than about 1 pound force(4.45 N). The forces exerted on the fluid transfer device as it is beingwithdrawn from a collection device 10 should be minimized to avoidstripping the fluid transfer device from, for example, the mountingprobe of a vacuum pipette. Insertion and withdrawal forces can bedetermined using conventional force measurement instruments, such as amotorized test stand (Model No. TCD 200) and digital force gauge (ModelNo. DFGS-50 ) available from John Chatillon & Sons, Inc. of Greensboro,N.C.

A cap 30A-E according to the present invention is generally provided incombination with a fluid-holding vessel 20 as components of a collectiondevice 10. The cap 30A-E and vessel 20 of the collection device 10 canbe joined by means of mated threads which allow the cap to be screwed,snapped or otherwise frictionally fitted onto an outer surface 21 of theside wall 22 at the open end of the vessel. When the cap 30A-E isfrictionally fitted onto the vessel 20, the bottom surface 37 of theside wall 35 of the cap is preferably in contact with the annular topsurface 23 of the vessel to provide an interference fit, therebyfacilitating the essentially leak-proof seal discussed above. The cap30A-E can be modified to further improve resistance of the collectiondevice 10 to leaking by providing an annular seal bead 58 to an outersurface 59 of the skirt 41, as shown in FIG. 4. If the seal bead 58 isincluded, it should be sized so that it will be in frictional contactwith the inner surface 24 of the side wall 22 of the vessel 20 but willnot substantially interfere with joining of the cap 30A-E and vessel.The annular center 60 of the preferred seal bead 58 is about 0.071inches (1.80 mm) from the bottom surface 37 of the side wall 35 andextends radially outwardly about 0.0085 inches (0.216 mm) from the outersurface 59 of the skirt 41, where the thickness of the skirt 41 abovethe seal bead 58 is about 0.052 inches (1.32 mm). The skirt 41preferably includes a beveled base 61 below the seal bead 58 tofacilitate joining of the cap 30A-E and vessel 20. In a preferredembodiment, the skirt 41 extends downward a vertical distance of about0.109 inches (2.77 mm) from the annular center 60 of the seal bead 58 tothe bottom surface 47 of the skirt and uniformly decreases in thicknessfrom about 0.0605 inches (1.54 mm) to about 0.025 inches (0.635 mm)between the annular center of the seal bead and the bottom surface ofthe skirt. The second bore 46 of this preferred embodiment has adiameter of about 0.340 inches (8.64 mm) and a height of about 0.218inches (5.54 mm).

When provided as a component of a kit, the collection device 10 of thepresent invention preferably includes a specimen retrieval device forobtaining a sample to be analyzed, where the specimen retrieval devicehas preferably been sized to fit within the interior space 11 of thecollection device after the cap 30A-E and vessel 20 have been joined. Apreferred specimen retrieval device is a swab, such as the swabdisclosed by Pestes et al., “Cell Collection Swab,” U.S. Pat. No.5,623,942. This particular swab is preferred because it is manufacturedto include a score line which is positioned on the stem of the swab,allowing the swab to be manually snapped in two after a specimen hasbeen obtained, leaving the lower, specimen-bearing portion of the swabentirely inside the vessel 20 component of the collection device 10.When the specimen is being transported to a clinical laboratory, thecollection device 10 also preferably includes a transport medium forpreserving the sample prior to analysis. Transport mediums are wellknown in the art and will vary depending upon the sample type andwhether cell lysis prior to analysis is necessary.

Additionally, a kit according to the present invention may includeinstructions recorded in a tangible form (e.g., contained on paper or anelectronic medium) which explain how the components of the collectiondevice 10 are to be manipulated when obtaining a fluid sample or how thecap 30A-E is to be secured onto the vessel 20 prior to transporting thecollection device to a clinical laboratory. Alternatively, or inaddition to, the instructions may detail proper pipetting techniques forretrieving at least a portion of the sample from the collection device10 prior to analysis. These instructions may include information abouttypes of fluid transfer devices that can be used to penetrate the cap30A-E, positioning of a fluid transfer device for penetrating the capand/or the amount of force needed to penetrate the cap. Theinstructional materials may also detail proper use of the collectiondevice when the sample is to be exposed to reagents and conditionsuseful for amplifying a nucleic acid sequence targeted for detection.

Amplification prior to detection is particularly desirable in diagnosticassays where the initial population of targeted nucleic acid sequencesin a sample is expected to be relatively small, making detection of thetargeted nucleic acid sequences more difficult. There are manyprocedures for amplifying nucleic acids which are well known in the art,including, but not limited to, the polymerase chain reaction (PCR),(see, e.g., Mullis, “Process for Amplifying, Detecting, and/or CloningNucleic Acid Sequences,” U.S. Pat. No. 4,683,195),transcription-mediated amplification (TMA), (see, e.g., Kacian et al.,“Nucleic Acid Sequence Amplification Methods,” U.S. Pat. No. 5,399,491),ligase chain reaction (LCR), (see, e.g., Birkenmeyer, “Amplification ofTarget Nucleic Acids Using Gap Filling Ligase Chain Reaction,” U.S. Pat.No. 5,427,930), and strand displacement amplification (SDA), (see, e.g.,Walker, “Strand Displacement Amplification,” U.S. Pat. No. 5,455,166).The particular reagents (e.g., enzymes and primers) and conditionsselected by practitioners will vary depending upon the particularnucleic acid sequence being targeted for detection and the specificamplification procedure to be followed. Those skilled in the art ofnucleic acid diagnostics, however, will be able to select appropriatereagents and conditions for amplifying a specific targeted nucleic acidsequence following a particular amplification procedure without havingto engage in undue experimentation.

While the present invention has been described and shown in considerabledetail with reference to certain preferred embodiments, those skilled inthe art will readily appreciate other embodiments of the presentinvention. Accordingly, the present invention is deemed to include allmodifications and variations encompassed within the spirit and scope ofthe following appended claims.

1. A method for removing a fluid substance from a closed systemcomprising a penetrable cap in sealing engagement with an open end of afluid-holding vessel, the method comprising the steps of: a) entering aninterior space defined by the cap and vessel of the system, wherein thestep of entering the interior space of the system comprises: i)penetrating a first frangible seal affixed to the cap with a fluidtransfer device such that air passageways are formed between the fluidtransfer device and the first frangible seal; and ii) penetrating asecond frangible seal affixed to the cap and axially aligned below thefirst frangible seal with the fluid transfer device such that airpassageways are formed between the fluid transfer device and the secondfrangible seal, thereby permitting air to be vented from the interiorspace of the system; b) drawing at least a portion of the fluidsubstance contained in the vessel into the fluid transfer device; and c)removing the fluid transfer device from the system.
 2. The method ofclaim 1 further comprising the step of passing the fluid transfer devicethrough a filter contained within the cap and interposed between thefirst and second frangible seals.
 3. The method of claim 2, wherein thefilter comprises a material selected from the group consisting of a pilefabric, a sponge, a foam, a felt, a sliver knit, a GORE-TEX ® fabric,and a fabric containing a LYCRA® fiber.
 4. The method of claim 3,wherein the filter comprises a pile fabric.
 5. The method of claim 1,wherein each of the first and second frangible seals comprises a foil.6. The method of claim 5 further comprising the step of passing thefluid transfer device through a filter contained within the cap andinterposed between the first and second frangible seals.
 7. The methodof claim 6, wherein the filter comprises a material selected from thegroup consisting of a pile fabric, a sponge, a foam, a felt, a sliverknit, a GORE-TEX® fabric, and a fabric containing a LYCRA® fiber.
 8. Themethod of claim 7, wherein the filter comprises a pile fabric.
 9. Themethod of claim 2, wherein the fluid transfer device is a plasticpipette tip.
 10. The method of claim 9, wherein step a) requires aninsertion force of less than about 8 pounds force and step c) requires awithdrawal force of less than about 4 pounds force.
 11. The method ofclaim 9, wherein step a) requires an insertion force of less than about6.5 pounds force and step c) requires a withdrawal force of less thanabout 3 pounds force.
 12. The method of claim 9, wherein step a)requires an insertion force of less than about 5 pounds force and stepc) requires a withdrawal force of less than about 2 pounds force. 13.The method of claim 9, wherein step a) requires an insertion force ofless than about 4.5 pounds force and step c) requires a withdrawal forceof less than about 1 pound force.
 14. The method of claim 1 furthercomprising the step of exposing at least a portion of the fluidsubstance removed from the system in step c) to nucleic acidamplification reagents and conditions.
 15. The method of claim 1,wherein steps a) - c) are performed by an automated instrument.
 16. Amethod for depositing a substance in a closed system comprising apenetrable cap in sealing engagement with an open end of asubstance-holding vessel, the method comprising the steps of: a)entering an interior space defined by the cap and vessel of the system,wherein the step of entering the interior space of the system comprises:i) penetrating a first frangible seal affixed to the cap with asubstance transfer device; and ii) penetrating a second frangible sealaffixed to the cap and axially aligned below the first frangible sealwith the substance transfer device; b) depositing one or more substancesin the vessel; and c) removing the substance transfer device from thesystem.
 17. The method of claim 16 further comprising the step ofpassing the substance transfer device through a filter contained withinthe cap and interposed between the first and second frangible seals. 18.The method of claim 17, wherein the filter comprises a material selectedfrom the group consisting of a pile fabric, a sponge, a foam, a felt, asliver knit, a GORE-TEX® fabric, and a fabric containing a LYCRA® fiber.19. The method of claim 18, wherein the filter comprises a pile fabric.20. The method of claim 16, wherein each of the first and secondfrangible seals comprises a foil.
 21. The method of claim 20 furthercomprising the step of passing the substance transfer device through afilter contained within the cap and interposed between the first andsecond frangible seals.
 22. The method of claim 21, wherein the filtercomprises a material selected from the group consisting of a pilefabric, a sponge, a foam, a felt, a sliver knit, a GORE-TEX® fabric, anda fabric containing a LYCRA® fiber.
 23. The method of claim 22, whereinthe filter comprises a pile fabric.
 24. The method of claim 13, whereinthe substance is a fluid substance.
 25. The method of claim 24, whereinthe fluid substance is a reagent for performing a diagnostic assay. 26.The method of claim 13, wherein steps a) - c) are performed by anautomated instrument.